Blattella germanica. Sexually mature females possess more C29 CHCs, especially the contact sex pheromone precursor 3,11-DimeC29. RNA interference (RNAi) screen against the fatty acid elongase family members combined with heterologous expression of the genes in yeast revealed that both BgElo12 and BgElo24 were involved in hydrocarbon (HC) production, but BgElo24 is of wide catalytic activities and is able to provide substrates for BgElo12, and only the female-enriched BgElo12 is responsible for sustaining female-specific HC profile. Repressing BgElo12 masculinized the female CHC profile, decreased contact sex pheromone level, and consequently reduced the sexual attractiveness of female cockroaches. Moreover, the asymmetric expression of BgElo12 between the sexes is modulated by sex differentiation cascade. Specifically, male-specific BgDsx represses the transcription of BgElo12 in males, while BgTra is able to remove this effect in females. Our study reveals a novel molecular mechanism responsible for the formation of SDHCs and also provide evidences on shaping of the SDHCs by sexual selection, as females use them to generate high levels of contact sex pheromone.Insect cuticular hydrocarbons (CHCs) serve as important intersexual signaling chemicals and generally show variation between the sexes, but little is known about the generation of sexually dimorphic hydrocarbons (SDHCs) in insects. In this study, we report the molecular mechanism and biological significance that underlie the generation of SDHC in the German cockroach  Sexual dimorphism of body waxes is prevalent in insects; this study reveals that the sex-differentiation pathway regulates fatty acid elongation, ensuring production of the sexually dimorphic cuticular hydrocarbons needed for high levels of sex pheromone and sexual attractiveness in female cockroaches. Transformer and Doublesex in insects) and the patterns of splicing these genes into sex-based isoforms have been widely described in different insect orders [Sexual dimorphism is prevalent in the animal kingdom. Females and males independently evolve some traits that enhance survival and reproduction under the pressure of divergent selection forces, thus leading to sexual dimorphism of traits such as body size . Under st orders –17. Howetrans-enoyl-CoA-reductase to yield the elongated VLCF acyl-CoA [The main function of insect cuticular hydrocarbons (CHCs) is to waterproof the cuticle to resist dehydration under dry conditions . In manyacyl-CoA ,33. The acyl-CoA –36. It iacyl-CoA –39. ThesDrosophila melanogaster, several studies have elucidated the pheromonal sexual dimorphism of CHCs. Female flies produce C27 and C29 dienes , both of which function as female-specific contact sex pheromone components, whereas male flies produce C23 and C25 monoenes (7-tricosene and 7-pentacosene) [Transformer, in male oenocytes feminized the male CHC profile and elicited homosexual courtship from other males [desatF was responsible for the generation of pheromonal dienes in female D. melanogaster [eloF [desatF in D. melanogaster females was due to a special cis-regulatory element (CRE) located upstream of desatF in D. melanogaster; the CRE presented a doublesex protein  binding site that could be recognized by the female-specific isoform of Dsx (Dsx-F), and the binding of Dsx-F activated the transcription of desatF, whereas male-specific Dsx showed no regulatory activity, resulting in sexual dimorphism of desatF expression [D. melanogaster, and studies in other insects are even rarer.In the fruit fly acosene) . Ferveurer males . These fnogaster , and theer [eloF . The spepression . HoweverBlattella germanica is a significant worldwide indoor pest [B. germanica [B. germanica function as waterproofing agents [B. germanica. BgTra is only functional in females and can splice BgDsx into 2 nonfunctional female-specific isoforms (BgDsxF), whereas males generate a complete functional male-type BgDsx (BgDsxM) [B. germanica.The German cockroach oor pest . Consideermanica ,44–46. Cg agents , and as g agents . The cong agents –51. More(BgDsxM) . These fB. germanica as a model insect. We characterize the SDCHCs, describe their temporal development, identify BgElo genes, and provide evidence that BgElo12 is responsible for the formation of SDCHC profiles. In addition, we found that the female-enriched HCs are important in the generation of the female-specific contact sex pheromone; RNA interference (RNAi) of BgElo12 in females decreased courtship responses of males. Moreover, we show that BgElo12 is under the regulation of the sex differentiation pathway; BgDsxM can specifically repress the transcription of BgElo12. These findings suggest that the generation of SDHCs is achieved by placing a fatty acid elongation gene (BgElo12) under the regulation of BgDsxM and linking the sex differentiation regulatory cascade with the HC biosynthesis pathway, resulting in the asymmetric gene expression and SDHCs in B. germanica.In this study, we employed BgFas1) and a P450 oxidative decarbonylase gene (CYP4G19) that are involved in HC biosynthesis, but both showed no function in the formation of sexual dimorphism of CHCs [BgElo genes in B. germanica.The temporal development of SDCHCs is rarely reported. In order to understand the molecular mechanisms of SDHC generation, we first analyzed the CHC profiles during sexual maturation. The oocytes of female cockroaches mature after eclosion by taking up vitellogenin until ovulation , and fem of CHCs ,56. HoweBgElo genes in the B. germanica genomic data and our full-length transcriptomic data [BgElo candidate genes were identified, and all BgElos were cloned and re-sequenced. Sequence alignment revealed that different BgElo proteins showed high homology and contained the conserved HXXHH and YXYY motifs [We found that the differences of CHCs between females and males are largely reflected in carbon chain length. Based on this assumption, we searched for potential mic data ,57. A toY motifs . All BgEBgElos in the fat body and abdominal integument. Results showed that BgElo24 and BgElo12 were highly expressed in the integument, and BgElo1, 2, 3, 6, 7, 9, 10, 11, 14, 17, 20, and 22 were also abundant in the integument, while other BgElos were nearly undetectable  was performed in early fifth-instar nymphs (N5D1 or N5D2), and a boost injection was performed 1 week later, and then the treated cockroaches were collected at different adult stages and subjected to CHC analysis. Knockdown of different NA level . Gas chrC27 CHCs . C28 CHCC28 CHCs . C29 CHCr amount . KnockdoC30 CHCs . CHCs wio24-RNAi . The detBgElo12 and BgElo24 are involved in HC production, it is unclear which gene is responsible for sexual dimorphism of CHCs. Therefore, we compared the CHC profiles of treated female cockroaches with wild-type male cockroaches. The results showed that only RNAi of BgElo12 made the female CHC profile more similar to the male profile  and early adult stage, but its expression level was higher in females than in males at AD3, and the difference increased through AD6 . The genetic background of S. cerevisiae is relatively clear, as wild-type S. cerevisiae contains 3 different ELO proteins: ELO1 is able to elongate C14 fatty acids (FAs) to C16 FAs, ELO2 can generate C24 FAs, and ELO3 plays essential roles in the conversion of C24 FAs to C26 FAs [S. cerevisiae that contained pYES2-GFP (control) detected large amounts of C16:1, C16:0, C18:1, and C18:0 FAs, minor amounts of C20 to C24 and C28 FAs, and a larger amount of C26:0 FAs  in ermanica , we suspractions .BgElo12-RNAi repression of pheromone production in females affected male courtship behavior. When the female contact sex pheromone is detected by a male, it displays a characteristic male courtship behavior, including wing raising (WR) [BgElo12 resulted in only a slight decline in WR rate (about 20%), but the latency of WR was significantly increased  . When anncreased . For usi seconds . These rBgElo12 in females, but the regulators that govern sexually dimorphic expression of BgElo12 in females and males are unknown. Wexler and colleagues reported that RNAi of the sex determination gene BgTra in females converts its CHC profile to a male-like profile [BgElo12 expression. We first confirmed the function of BgTra and BgDsx in sex-specific development via RNAi. For each gene, we used 2 unique RNAi targets within a conserved sequence region for multiple isoforms. RNAi of BgTra and BgDsx in females or males showed similar phenotypes as described by Wexler and colleagues [BgTra is only functional in females, while BgDsx only works in males  primers that target all isoforms of each gene. The expression of both BgTra and BgDsx increased after eclosion, but BgTra transcript levels tended to be stable from AD4 to AD6  [BgElo12 in females after RNAi of BgTra was caused by the generation of BgDsxM. This hypothesis was supported by co-injection of dsBgTra and dsBgDsx and a dual-luciferase reporter assay that BgDsxM could directly modulate the expression of BgElo12 by interaction with its upstream sequence region  , we suspe region . Further BgElo24 , suggestBgTra and BgDsx on HC profiles. Knockdown of BgTra in females masculinized the CHC profile and increased the male-enriched CHC components; however, this change disappeared by co-injection of dsBgTra and dsBgDsx . Overall, our results indicate that the sexual dimorphism of HCs in B. germanica is primarily determined by BgElo12, and sex determination pathway genes are the critical regulators that control the asymmetric expression of BgElo12 between males and females.We further analyzed the effects of Dsx Figs , S11A. Snts Figs , S11B. IB. germanica. The CHC profiles in insects are regulated by complex biosynthetic and transport pathways, involving multiple gene families. We demonstrated that the fatty acid elongation step is responsible for sexual dimorphism of CHCs in B. germanica, We identified that both BgElo12 and BgElo24 were participated in HC biosynthesis, but only the female-enriched BgElo12 is the core gene that encodes for the elongase involved in generating more female-enriched HCs, and the asymmetric expression of BgElo12 between the sexes is modulated by sex differentiation genes: BgDsxM represses the expression of BgElo12 in males, while BgTra removes this repression in females , but it did not affect the male-enriched peak 17 , which generated a male-like HC profile; however, RNAi of BgElo24 unselectively down-regulated all C28 to C30 HCs, indicating that only BgElo12 was involved in the formation of SDHCs in B. germanica. Lastly, we found both RNAi of BgElo12 and BgElo24 increased some C27 HCs, indicating that there are some other BgElos involved in HC production. The identification of these genes will be arduous, because the C27 compounds occur in small amounts, and multiple BgElos may catalyze the synthesis of the same HC independently. It is worth noting in this regard that Blattella asahinai, a sister species of B. germanica produces almost no C27 compounds [Studies of the genetic bases of HC biosynthesis in insects have demonstrated that chain lengths are determined by the fatty acid elongation process: The rate-limiting enzyme elongase determines the chain lengths of VLCFA products and produces the final HCs with different chain lengths . In our than C28 . BgElo24ompounds . These 2B. germanica, as in other insects, HCs are transported through the hemolymph and selectively incorporated into or deposited on various tissues, including the cuticle, ovaries, and specialized pheromone glands [BgElo12-RNAi on internal HCs demonstrated that the female- and male-specific CHC profiles were not caused by selective transport, but rather by differences in de novo HC biosynthesis between the sexes, regulated by BgElo12. Also, the total amount of internal HCs was significantly decreased in BgTra-RNAi females, while it was increased in BgDsx-RNAi males  family of transcription factors includes conserved developmental regulators in the sex differentiation pathway, governing the fate of sexually dimorphic traits in animals [Dsx in arthropods, which is related to Dmrt, works through sex-specific splice variants that are controlled by Tra in many insects. Sex-specific Dsx isoforms promote sexual differentiation by modulating diverse downstream genes; thus, the Dsx gene is regarded as a central nexus in sexual differentiation [BgTra converted the female cockroach CHC profiles to male-like profiles [BgTra in female cockroaches indeed down-regulated the expression of BgElo12, but the effect of BgTra-RNAi on BgElo12 expression could be recovered by co-injection of dsBgTra and dsBgDsx, and RNAi of BgDsx in males up-regulated BgElo12 transcripts. These results suggest that BgDsxM can repress the transcription of BgElo12 in males, and the down-regulation of BgElo12 by repressing BgTra was caused by the conversion of BgDsxF to BgDsxM, as BgTra is able to regulate the splicing of BgDsxM to BgDsxF [BgElo12 by BgDsx connects the sex differentiation pathway with the HC biosynthesis pathway, and, therefore, enables the sexual dimorphism of HCs in B. germanica.In this study, we first demonstrated that  animals . Dsx in ntiation ,69. Prevprofiles . In our o BgDsxF . The regBgDsxM can directly regulate the transcript of BgElo12, there may be other indirect regulatory pathways. Several candidate factors have been shown to regulate the HC profiles in various insects , including ecdysone, juvenile hormone (JH), biogenic amines, and the insulin signaling pathway [M. domestica [BgDsx and BgElo12. However, more detailed investigations are needed to thoroughly elucidate the differential regulation of HC production in both females and males.Although the dual-luciferase reporter gene assay suggested that  pathway ,70–77. Iomestica . Ecdysonomestica ,79, and omestica . TherefoDsx also regulates the HC biosynthesis pathway in D. melanogaster; DsxF specifically activates the transcription of desatF and generates pheromonal dialkenes [BgDsxM suppresses the generation of female traits in male cockroaches, while BgTra removes the inhibitory effect of BgDsxM in females [BgDsxM represses the expression of BgElo12, and, therefore, it represses the generation of female-enriched HCs in males, and BgTra is crucial in maintaining female-enriched HCs, especially for the contact sex pheromone precursors. However, BgTra may also affect contact sex pheromone biosynthesis in female cockroaches through additional pathways.ialkenes . However females . Thus, BB. germanica is clearly subject to both natural and sexual selection because it is derived from a prominent CHC, which also serves as a waterproofing component of the CHC profile and is maternally invested in offspring [B. germanica, where the pheromone and its HC precursor serve in sexual communication, resilience to environmental stressors, and in maternal investment in eggs.Sexually dimorphic traits are generated in females and males in response to intra- and intersexual selection, but pleiotropic traits are also subject to natural selection, especially when they are also shaped by and adapted to environmental stresses ,81. Mostffspring . Thus, mffspring . This asDrosophila. In B. germanica, as well, male cockroaches have a unique CHC profile, especially enriched in C27 components with 9-; 11-; 13-; and 15-MeC29 being particularly prominent. The male-specific HC profile is generated during sexual maturation, suggesting that it may function in sexual communication. It is possible that the male-enriched 9-; 11-; 13-; and 15-MeC29 may function as a sex pheromone in 3 related contexts: (a) it may distinguish males and females within cockroach aggregations; (b) it may signal “maleness” and male quality to females; and (c) it may function in male–male recognition, contests, and competition for access to females. Moreover, it is possible that male-specific P450s may catalyze the oxidation of these male-enriched HCs to homologous methyl ketones, as in females, and, in turn, serve these functions. However, more bioassays are required to analyze the biological significance of male-specific CHC profiles.In systems that use CHCs in sexual communication, it is common for the CHC profiles to contain female- and male-specific components, as is evident in B. germanica originated from a laboratory strain collected in the 1970s. The cockroaches were maintained in aquaria at 30 ± 1°C with a relative humidity (RH) of approximately 50% under 12:12 hour light–dark photoperiod regime and fed rat chow and tap water. Newly hatched cockroaches were separated and reared in new containers. Early-stage (day 1 or 2) fourth- and fifth-instar nymphs were separated and used in dsRNA injection; newly emerged adults were collected and reared in plastic jars for experiments.The German cockroach B. germanica cuticular lipids were extracted as described [n-Hexacosane (15 μg) or 14-heptacosanone (0.5 μg) were added as internal standards. The extracts were combined and reduced to approximately 300 μL with a nitrogen flow and loaded onto a Pasteur pipette silica gel mini-column, as previously described [n-hexacosane and 1 μg of 14-heptacosanone were added for quantification of internal HCs and methyl ketones, respectively. The homogenate was vigorously vortexed and centrifuged at 2,500 g for 10 minutes. The supernatant hexane phase was collected, and the extraction was repeated using n-hexane. Separation of HCs and methyl ketones was performed using column chromatography, as described above.escribed . Individescribed . The CHCescribed . Each suLipid analysis was performed with a TRACE 1310 GC–ISQ single quadrupole MS . In brief, lipids were separated on a DB-5MS capillary column . The oven started at 60°C and kept for 2 minutes, heated to 160°C for HCs and methyl ketones, or 220°C for fatty acid methyl esters (FAMEs) at a rate of 30°C/min, then increased at 3°C/min up to 250°C, followed by 10°C/min up to 320°C and held for 5 minutes. Electron ionization mode (70 eV) was used, and the MS scan range was 45 to 650 m/z at a rate of 5 scans/s. Identification of HC compounds was performed according to Jurenka and colleagues , and peaactin5c (GenBank: AJ862721.1) and calculated using the 2−ΔΔCt method. Each treatment contained 4 biological replicates and technical triplicates.Total RNA was isolated with RNAiso Plus Reagents  according to the manufacturer’s instructions. cDNA was reverse-transcribed from 800 ng of total RNA using the PrimeScrip RT reagent Kit with gDNA Eraser (Takara). Gene-specific primers with appropriate amplification efficiency (0.95 to 1.05) were screened by a cDNA dilution series . QuantifBgElo genes in B. germanica genome data [Elongase from D. melanogaster as query sequences. Candidate BgElo genes were amplified with the PrimeSTAR GXL DNA Polymerase reagent (Takara) (primers are listed in BgElo genes were then translated and submitted to SMART online tools (http://smart.embl-heidelberg.de) to analyze the conserved structures; only genes with the typical ELO domain were confirmed as BgElo genes. The putative BgElo mRNA sequences were mapped to the genomic data (GenBank: PYGN00000000.1) using a local BLASTN tool, and the intron–exon structure was analyzed based on the GT–AG rule. The conservative motifs in BgElos were analyzed by sequence alignment with DNAMAN 9.0 software.Both BLASTN and BLASTP were used to search ome data  and our BgElo genes involved in HC biosynthesis, the expression levels of different BgElo genes were quantified in the fat body and abdominal integument, where HCs or their precursors were generated. In addition, other tissues including the head, thorax, gut, legs, ovaries, Malpighian tubules, ejaculatory duct , and colleterial glands were dissected from 2-day-old females and were used to analyze the expression profiles of BgElo12 and BgElo24 among different tissues. In order to study the time course of BgElo12, BgElo24, BgDTra, and BgDsx transcript levels during sexual maturation, a representative nymphal stage  and AD0 to AD6 (0-day-old to 6-day-old adult) females and males were collected. Total RNA was extracted from various tissues or intact cockroaches, and the expression profiles of different genes were studied via RT-qPCR.In order to screen the potential Mus musculus (Muslta) used for dsRNA synthesis were amplified and cloned into pMD 19-T Vector (Takara). Templates used for single-stranded RNA were amplified with primers that incorporated with the T7 promoter sequence  with a dosage of 3 μg in 2 μL; the second injection was performed 1 week later with a dosage of 4 μg in 2 μL. For confirming the function of BgElo12 in contact sex pheromone biosynthesis, a third injection with 4 μg of dsRNA in 2 μL was carried out on , the second at early fifth instar (N5D1-N5D2), and the last at early sixth instar (N6D1 to N6D2). The fourth-instar cockroaches were injected with approximately 0.5 μg of dsRNA in approximately 0.2 μL; the fifth- and sixth-instar cockroaches were injected with 1 μg of dsRNA in 2 μL. In order to verify the function of BgElo12 and BgElo24 in HC biosynthesis and sex determination genes in modulating BgElo12 mRNA levels or HC profiles, 2 nonoverlapping gene-specific targets were designed and used in this study; as there are some isoforms of BgDsx and BgTra, the RNAi targets were designed within the common sequence region. After dsRNA injection, intact AD2 cockroaches were used for RNAi efficiency analysis.Gene-specific target sequences as well as a heterologous fragment from sequence . DiffereBgElo12, BgElo24, or control (GFP) were amplified with PrimeSTAR HS DNA Polymerase (Takara) using gene-specific primers  and the yeast consensus sequence (TACACA) following the restriction enzyme sites (only for forward primers). The amplified BgElo12, BgElo24, or GFP CDS fragments were ligated into the linearized pYES2 shuttle plasmid (Thermo Fisher Scientific) and verified by sequencing. The recombinant plasmids were transformed into INVSc1 S. cerevisiae (Thermo Fisher Scientific) using the PEG-LiAc method and streaked onto S. cerevisiae minimal medium minus uracil (SC–uracil) plates to select transformants; single colonies were inoculated in SC–uracil medium with 2% glucose. After culturing at 30°C for 24 hours, the yeast was collected and diluted to an OD600 of 0.4 with SC–uracil medium containing 1% raffinose and 2% galactose and further cultured at 30°C until they reached an OD600 of 0.8. At this point, transcription of exogenous genes was examined by real-time PCR (RT-PCR). Substrates of C20 (0.5 mM), C22 (1 mM), C24 (1 mM), C26 (1 mM), C28 (1 mM), 2-methylhexadecanoic acid (0.5 mM), and 14-methylhexadecanoic acid (0.5 mM) were separately added into the cultures with an extra 1% of tergitol type Nonidet P-40. All the substrates were purchased from Sigma-Aldrich  or TCI .Heterologous expression was performed as described . Complet primers  that con2SO4 in methanol was added, and the mixture was vortexed and incubated at 80°C in a N2 atmosphere for 2 hours [After 48 hours, yeast cells were harvested by centrifugation at 500 g for 5 minutes and washed thrice with Hanks’ balanced salt solution for fatty acid derivatization and analysis. Pellets were dried under a steam of nitrogen, and 2 mL of 1% (v/v) H 2 hours . After tBgElo12 and BgElo24 to contribute to water retention was assessed by a desiccation bioassay. Drying bottles were prepared by putting approximately 120 g of packed fresh silica gel into an approximately 900-mL sealed plastic bottle. The RH inside the bottle dropped to 5% within 2 hours, which was monitored by HOBO Pro v2 . AD2 females were injected with dsBgElo12, dsBgELo24, and dsMuslta and separately caged in the desiccation bottles at 30°C, supplied with approximately 1 g of dry food, but no water. Survival was recorded every 8 hours until all the cockroaches died. About 100 cockroaches were used for each treatment.The capacity of Courtship behavior was tested as described ,87. We fBgElo12 was obtained by 5′ Rapid Amplification of cDNA Ends (RACE). The 5′ RACE cDNA library was prepared using Clontech SMARTer RACE 5’/3’ Kit (Takara) according to the user manual with the gene-specific primer  were separately cloned into the expression vector pCDNA3.1. The HEK293T cells were cultured in a 24-well plate with 500 μL of Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific) for 24 hours before transfection, and the restructured pGL3-basic vector (200 ng/well) was co-transfected with the expression vectors (200 ng/well) to HEK293T cells using Lipofectamine 3000 . The pRL-TK that encoded a Renilla luciferase was also co-transfected as an internal control. The transfected cells were cultured at 37°C for 36 hours and subjected to luciferase activity analysis using the Dual-Glo Luciferase Assay System (Promega).The 5′ end of c primer  and kit-t test was used for 2-group comparison; significant differences between multigroups were analyzed by 1-way ANOVA followed by the least significant difference (LSD) test  or Welch ANOVA followed by Games–Howell multiple comparisons test  at P < 0.05 level. PCA was used to distinguish the CHC profiles of AD1 and AD6 cockroaches.Data were statistically analyzed using SPSS 23 and presented as mean ± SEM or mean ± SD. Two-tailed Student S1 FigPeak 24 represents the female-enriched 3,7-; 3,9-; and 3,11-DimeC29, and Peak 17 is the male-enriched 9-; 11-; 13-; and 15-MeC29. The data underlying this figure are included in (TIF)Click here for additional data file.S2 FigP < 0.01, 2-tailed Student t test. The data underlying this figure are included in Data are shown as mean ± SEM, calculated from 3 to 4 replicates (2–3 cockroaches/replicate); **(TIF)Click here for additional data file.S3 FigP < 0.05). The data underlying this figure are included in Different letters indicate significant differences between groups using Welch ANOVA Click here for additional data file.S4 Fig Relative amount of individual CHCs in males after knockdown of BgElo12 and BgElo24. Data are shown as mean ± SEM; *P < 0.05, **P < 0.01; 2-tailed Student t test, n = 9 or 10. (C) PCA of CHC profiles after repression of BgElo12 and BgElo24; each dot represents a datum calculated from one cockroach. (D) The corresponding loading diagram; the numeric sequence labels correspond to numbers and CHC components in (TIF)Click here for additional data file.S5 Fig(A) Analysis of CHCs after RNAi of BgElo12 using the second target (dsBgElo12B) (B) or after RNAi of BgElo24 using the second target (dsBgElo24B).  The effects of BgElo12-RNAi and BgElo24-RNAi on internal HCs. Data are shown as mean ± SEM; *P < 0.05, **P < 0.01; 2-tailed Student t test, n = 9–12. The data underlying this figure are included in (TIF)Click here for additional data file.S6 FigP < 0.05), n = 4. The data underlying this figure are included in Data are shown as mean ± SEM; and each sample was collected from 4 , 8 , and 12 (MT) cockroaches. Different letters indicate significant differences between groups using Welch ANOVA Click here for additional data file.S7 FigBgElo24 up-regulated the expression of BgElo12(A), while RNAi of BgElo12 did not affect BgElo24 transcript level (B). Data are shown as mean ± SEM; P values were calculated from 4 samples; each sample contained 2 cockroaches; 2-tailed Student t test. (C) Detection of GFP protein in the yeast with pYES2-GFP using a FV3000 confocal fluorescence microscope (Olympus).  RT-PCR analysis of the BgElo12 and BgElo24 mRNA after the induction with galactose.  Gas chromatograms of FAMEs after adding 2-MeC16:0 into the medium.  Gas chromatogram of FAMEs after adding 14-MeC16:0 into the medium. The compositions with retention times between 12 and 28 minutes were magnified about 50 times.  Mass spectra of methyl octacosanoate and methyl triacontanate, both of which showed a strong characteristic ion fragment (m/z = 74) and M peak. The data underlying RNAi of (TIF)Click here for additional data file.S8 FigBgTra in females generated a male-like body size and cuticle color, male-like tergal gland structure, and a protruding tissue at the end of the abdomen (left center); RNAi of BgDsx in males generated a female-like body color and a protruding tissue at the end of the abdomen, and the tergal gland partly disappeared (right bottom). Other treatments did not generate obvious developmental effects. RNAi, RNA interference.RNAi of (TIF)Click here for additional data file.S9 FigP values were calculated from 12 replicates; 2-tailed Student t test. The data underlying this figure are included in Data are shown as mean ± SEM; (TIF)Click here for additional data file.S10 FigP values were calculated from 4 replicates (2 cockroaches/replicate); 2-tailed Student t test. The data underlying this figure are included in Data are shown as mean ± SEM; (TIF)Click here for additional data file.S11 FigP < 0.05, **P < 0.01, 2-tailed Student t test, n = 14 , 12 , 14 , 12 , and 16 . The data underlying this figure are included in Data are shown as mean ± SEM, *(TIF)Click here for additional data file.S12 Fig Effects of BgTra-RNAi in females and BgDsx-RNAi in males on sex-specific internal HC profiles.  Proportion changes of representative internal HCs after RNAi of BgTra in females and BgDsx in males.  Effects of BgTra-RNAi in females and BgDsx-RNAi in males on total amounts of internal HCs. Data are shown as mean ± SEM, *P < 0.05, **P < 0.01, 2-tailed Student t test, n = 10 or 11. The data underlying this figure are included in (TIF)Click here for additional data file.S1 TableCHC, cuticular hydrocarbon.(DOCX)Click here for additional data file.S2 TableCHC, cuticular hydrocarbon; RNAi, RNA interference.(DOCX)Click here for additional data file.S3 TableFAME, fatty acid methyl ester.(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file. 1 Apr 2021Dear Dr Liu, Thank you for submitting your manuscript entitled "Modulation of fatty acid elongation sustains sexually dimorphic hydrocarbons and female attractiveness in Blattella germanica (L.)" for consideration as a Research Article by PLOS Biology.Your manuscript has now been evaluated by the PLOS Biology editorial staff, as well as by an academic editor with relevant expertise, and I'm writing to let you know that we would like to send your submission out for external peer review.However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. 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Given the disruptions resulting from the ongoing COVID-19 pandemic, please expect delays in the editorial process. We apologise in advance for any inconvenience caused and will do our best to minimize impact as far as possible.plosbiology@plos.org if you have any queries relating to your submission.Feel free to email us at Kind regards,Roli RobertsRoland G Roberts, PhD,Senior EditorPLOS Biology 26 May 2021Dear Dr Liu,Thank you very much for submitting your manuscript "Modulation of fatty acid elongation sustains sexually dimorphic hydrocarbons and female attractiveness in Blattella germanica (L.)" for consideration as a Research Article at PLOS Biology. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by four independent reviewers.You'll see that all of the reviewers are positive about your study, and only have a few requests . In light of the reviews (below), we are pleased to offer you the opportunity to address the comments from the reviewers in a revised version that we anticipate should not take you very long. We will then assess your revised manuscript and your response to the reviewers' comments and we may consult the reviewers again.IMPORTANT:a) Please change your title to "Modulation of fatty acid elongation in cockroaches sustains sexually dimorphic hydrocarbons and female attractiveness"b) Please provide a blurb, according to the instructions in the submission form.c) When revising your manuscript, please bear in mind the need to make the paper accessible to our broad readership .d) Please attend to all of the requests from the reviewers.e) Please attend to my Data Policy requests further down. Specifically, we note that you have provided some of the underlying data in the Supplementary Data files, but we think that data for some of the panels have not been presented separately. 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Please don't hesitate to contact us if you have any questions or comments.Sincerely,Roli RobertsRoland RobertsSenior EditorPLOS Biologyrroberts@plos.org*****************************************************DATA POLICY:http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797 You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:1) Supplementary files . Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to  using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).2) Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication. We note that you already present most of your underlying data in the supplementary data files S1 Data and S2 Data However, several panels are missing: Figs 1FG, 2CDEF, 3DE, 5CDEE’FF’, 6FG, S2, S3, S7AB, S10, S12CD. I suspect that these overlap with data presented for other panels, but please could you present it separately? NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived .IMPORTANT: Please also ensure that figure legends in your manuscript include information on where the underlying data can be found, and ensure your supplemental data file/s has a legend.Please ensure that your Data Statement in the submission system accurately describes where your data can be found. ------------------------------------------------------------------------BLOT AND GEL REPORTING REQUIREMENTS:https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements We require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare and upload them now. Please carefully read our guidelines for how to prepare and upload this data: ------------------------------------------------------------------------DATA NOT SHOWN?- Please note that per journal policy, we do not allow the mention of "data not shown", "personal communication", "manuscript in preparation" or other references to data that is not publicly available or contained within this manuscript. Please either remove mention of these data or provide figures presenting the results and the data underlying the figure(s).------------------------------------------------------------------------REVIEWERS' COMMENTS:Reviewer #1:This is a very well written manuscript that describes fatty acyl-CoA elongases and their regulation of elongation and pheromone production of hydrocarbon related pheromones in the German cockroach. Very little is known of the elongation process in producing hydrocarbons in insects, and this manuscript addresses this void. It is my opinion that it describes very careful and well done experiments.Minor suggestions. Line 25. Correct the spelling of /underling'.Line 70. I think that it would be useful to describe in a little more detail that the elongase they are looking at is the first step and rate limiting step in elongation, which is then followed by they three steps indicated, reduction of the ketone to an alcohol, dehydration of the alcohol to the alkene and then reduction of the alkene to a saturated fatty acyl-CoA.Line 422. ....only n-alkanes and... with the n italicized.Line 479. One of the best examples of hormonal regulation of chain length of hydrocarbons is the housefly, where ecdysone results in C27 alkenes being produced to where C23 alkenes become the dominant alkene. Reviewer #2:This paper addresses the biosynthetic pathway and genetic mechanisms controlling sexual differentiation of cuticular hydrocarbon profiles in the German cockroach, Blatella germanica. The authors characterize two elongases involved in cuticular hydrocarbon (CHC) production and identify one that appears to underlie sexually dimorphic profiles and is under control of the tra/ dsx sex determination pathway. Manipulation of the elongase, dsx, and tra shift the CHC profile and results in masculinization of female CHCs. Overall, a tremendous amount of work went into this manuscript. The CHC measurements and in-vitro characterization of the elongase activities are convincing. The authors show interesting behavioral and physiological consequences resulting from knockdown of the elongase. The CHC and gene expression quantitation is carefully done and the proposed pathway is supported by the data. Despite the overall excellence of the technical work, some of the data need to be reanalyzed with proper statistical tests and more details are needed about the methods. The significance of the findings needs to be more explicitly stated for a general science audience. The role of the sex determination pathway in sexually dimorphic morphological features is well-documented. Aside from Drosophila, are there other examples of the SD cascade in controlling CHCs or pheromones? This manuscript would be greatly improved with the editing of a native English speaker. In addition, if this article is meant for a broad audience, in its current form the text is rather dense. There are also some experiments and observations included that are somewhat tangential and should be omitted (details below)Minor corrections61: Actually, the enzymatic pathways are well-characterized in multiple insect orders, as evidenced by the references provided in the rest of the paragraph and multiple reviews . Authors should consider including a pathway illustrating the biosynthesis of HCs to accompany this portion of the introduction.74: replace "genes" with "enzymes". Again, I take issue with the statement that the genes/ enzymes are not well-studied. fact that the genes have been studied in few inspect species.99-103: section on courtship can be shortened since this is discussed in detail in the results section.172: "RNAi of the genes" is lab jargon. I suggest replacing "RNAi" with "knockdown"220: N6D4: Please define in the text. It is only explained in Fig 6 legend.228-229: awkwardly worded - "contributes to" in place of "support"? Also, the statement that "the role of Elo24 is desication tolerance" is overly general, based on a single assay. The enzyme may contribute to desiccation tolerance but it likely has other functions.246: It looks like HCs in the C27 - C32 range were downregulated, not just C28-29.255-256: Sentence could be reworded to make it more explicit what GFP expression indicates.308 - 317: This section could be shortened and the explanation of age used for assays can go in methods370-383: The observations about internal vs cuticular HC levels in this section are interesting but confusing in light of results showing that BgElo12 RNAi does not impact HC transport. The findings are also tangential to the main results and the underlying mechanisms can only be speculated upon at this point. I suggest drastically shortening this section, moving to the discussion section, or removing. 453-455: Sentence construction is awkward.478-479: The 20-HE steroid dehydrogenase was also identified as a regulator of HC profiles:Chiang YN, Tan KJ, Chung H, Lavrynenko O, Shevchenko A, Yew JY. Steroid Hormone Signaling Is Essential for Pheromone Production and Oenocyte Survival. PLoS Genet. 2016 Jun 22;12(6):e1006126. doi: 10.1371/journal.pgen.1006126. PMID: 27333054; PMCID: PMC4917198.488-489: Sentence construction is awkward.494-497: I suggest removing text related to krh1. If authors think this is an important result, it should go into results section. However, the data provided are not sufficient to support the conclusion in 498-499.MethodsMore details are needed on the following methods:1. How were the identifies of chromatogram peaks identified?2. How was quantitation of GCMS peaks performed? The numbers provided in S1 and S2 tables are presented as absolute quantities. However, absolute quantification would require comparing the intensity of the peaks to a synthetic version of the compound. It does not appear that synthetic standards were used. The tables and figures should indicate that "relative quantities" are measured rather than absolute amounts. More details are also needed as to how percentage  was calculated. Is it percentage of all peaks detected or just percentage of HCs? 3. Internal HC extractions: the fat body is diffuse throughout the body of the insect. How was it dissected in a standardized way from individual to individual? The reference  did not provide more details. Figures and TablesS1, S2 table: "ug" in the footnote should be replaced with "μg". Please add sample size to footnote. If these numbers were calculated based on the relative intensity of the compound to the internal standard, they are relative quantities rather than absolute quantities. Table title should be "Relative quantification…"S4, 5: See comment for Fig 3. S6 7A: What is the unit of y-axis? Fig 3B, C: An ANOVA is needed here to compare the relative amounts of individual CHCs rather than multiple t-tests. Before ANOVA can be performed, the CHCs should be collapsed into fewer categories so that the number of categories is less than the sample size. Y-axis should indicate that these are relative quantities.Fig 3D: comparison of each CHC type between 3 different groups requires an ANOVA rather than multiple t-tests. Fig 5A: Was the C29 methyl ketone quantified using a synthetic standard or do numbers represent abundance relative to internal standard? Fig 6F: ANOVA should be used to compare between the 3 genotypes. Reviewer #3:This study by Pei and co-workers addresses the interesting question of how sexually-dimorphic traits can emerge in animals, even though the genomes of males and females are essentially the same. In insects, cuticular hydrocarbons play a dual role. On the one hand, they protect from desiccation. On the other hand, they have in many cases been co-opted to play a role in chemical communication. Using the German cockroach, Blatella germanica, the authors unravel the molecular mechanism responsible for the formation of sexually-dimorphic cuticular hydrocarbons. Specifically, they identify elongase genes as major modulators of cuticular hydrocarbon composition. The study is thorough, from the characterization of sexually dimorphic cuticular hydrocarbons and their temporal development in both sexes to the identification of members of the multigene family encoding elongases . Furthermore, the authors elegantly explore the behavioral consequences of altering a single gene abundance via RNA interference. Finally, they built on previous work by Wexler et al  and begin to explore how the sex-differentiation genes Transformer and Doublesex act as upstream regulators of BgElo12. All in all, this is a very interesting and complete study, with nice illustrations and clearly-presented results and conclusions. I have only a few small comments. In the RNAi efficiency experiments, it is not clear which tissue was used to performed these experiments. Lines 185-186: the authors wrote "we cannot rule out that other BgElo genes might have a less prominent role in HC biosynthesis". I think it is necessary to specify that this statement applies to the very long chain hydrocarbons (C27-C30), to reflect the data presented in the paper. Figure 2: while there is nothing wrong with using the Games-Howell multiple comparisons test, I think it would be more appropriate to use Dunnet's procedure and test dsBgEloX treatment vs dsMuslta control. The conclusions would most certainly not be affected. Figure 3: It looks like knocking down Elo24 is leading to less CHCs overall. It might be useful to visualize the data using percentages (absolute quantities). Also, I think it may be helpful to provide a PCA with the different treatments. Similarly to what authors do in Fig 1, a PCA plot would help the readers see the similarities and differences between female and male profiles and the impact of the treatments. Reviewer #4:Pei et al. report the molecular mechanism underpinning the generation of sexually dimorphic hydrocarbons in the German cockroach. Expression analysis and RNAi screen against members of the fatty acid elongase gene family together with gene assays in a heterologous yeast system clearly demonstrated that both BgElo12 and BgElo24 are involved in the production of the hydrocarbons but only the female-enriched BgElo12 is responsible for sustaining the female-specific hydrocarbon profile. Furthermore, they reveal the involvement of two expression factors modulating the asymmetric expression of BgElo12 between the sexes.The impressive amount of work results in an almost TOO massive manuscript, including 88 references, 12 supplementary figures, 4 supplementary table, one appendix and two data supplements. My focus has been on the main text and its 7 figures.The study is very thorough, it addresses important scientific questions and the findings are of general interest. The manuscript is very well written and the conclusions supported by the results. The biosynthetic reaction under detailed study is the elongation of fatty acids, acids involved as precursors in the biosynthesis of the hydrocarbons produced from these fatty acids, hydrocarbons subsequently serving as precursors for the actual pheromone. - Can the authors explain and rectify why they did not analyze the effects of the elongases on the fatty acid profiles but only the resulting hydrocarbon profiles? - This appears especially important as they could not demonstrate the activity of BgElo12 and BgElo24 on the elongation of the methyl-branched fatty acids that have to be involved in the pheromone biosynthesis (cf. Lines 280-281). My additional few comments are all very minor:Line 25: The sentence starting "Here, we report…." Does not read well to me. English is not my native language but I wonder if "underling" is really the right word? I would suggest "underpins".Line 28: I would suggest that the sentence is slightly rephrased: "RNAi screen against the fatty acid elongase family members combined with heterologous expression of the genes in yeast revealed that both…."Line 412: Delete "were"?Line 505-506: While this is obviously true for moths (Lepidoptera) I wonder if it is generally true? Can the authors provide a reference?Fig 1: Black=Male and Red=Female seems to apply to all panels but G. I would suggest the authors consider changing the colour code in G and possibly move the Black/Red -Male/Female explanation out of the "Frame A" as it applies to all of the other panels.Fig 2: What is the unit on y-axis in A and B?Fig 3: How do I find out what the different peak numbers stand for in A, B, C, D and E? 13 Jun 2021AttachmentRespons to reviewers.docxSubmitted filename: Click here for additional data file. 18 Jun 2021Dear Dr Liu,On behalf of my colleagues and the Academic Editor, Richard Benton, I'm pleased to say that we can in principle offer to publish your Research Article "Modulation of fatty acid elongation in cockroaches sustains sexually dimorphic hydrocarbons and female attractiveness" in PLOS Biology, provided you address any remaining formatting and reporting issues. These will be detailed in an email that will follow this letter and that you will usually receive within 2-3 business days, during which time no action is required from you. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have made the required changes.IMPORTANT: The academic editor noticed that in Figure 3D, where you show "percentage changes", the y-axis runs from 0 to 1.0 ; if you really meant that the y-axis runs from 0 to 100%, then we recommend that you modify the labelling here .http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.Please take a minute to log into Editorial Manager at biologypress@plos.org. If you have not yet opted out of the early version process, we ask that you notify us immediately of any press plans so that we may do so on your behalf.PRESS: We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with http://www.plos.org/about/media-inquiries/embargo-policy/.We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit Thank you again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study. Sincerely,Roli RobertsRoland G Roberts, PhD Senior Editor PLOS Biologyrroberts@plos.org


C. elegans is an established model organism for studying genetic and drug effects on aging, many of which are conserved in humans. It is also an important model for basic research, and C. elegans pathologies is a new emerging field. Here we develop a proof-of-principal convolutional neural network-based platform to segment C. elegans and extract features that might be useful for lifespan prediction. We use a dataset of 734 worms tracked throughout their lifespan and classify worms into long-lived and short-lived. We designed WormNet - a convolutional neural network (CNN) to predict the worm lifespan class based on young adult images (day 1 – day 3 old adults) and showed that WormNet, as well as, InceptionV3 CNN can successfully classify lifespan. Based on U-Net architecture we develop HydraNet CNNs which allow segmenting worms accurately into anterior, mid-body and posterior parts. We combine HydraNet segmentation, WormNet prediction and the class activation map approach to determine the segments most important for lifespan classification. Such a tandem segmentation-classification approach shows the posterior part of the worm might be more important for classifying long-lived worms. Our approach can be useful for the acceleration of anti-aging drug discovery and for studying C. elegans pathologies. Caenorhabditis elegans (C. elegans) is an established model for studying various interventions into the aging process, which allowed to find numerous genes and drugs interfering with aging. 5 out of 7 Tier 1 and 4 out of 6 Tier 2 anti-aging drugs considered for human trials extend lifespan in the C. elegans model. There are many aging pathways conserved among species and the worms are expected to be used extensively not only in longevity research but also in the appearing anti-aging industry . Inte (28%) . Furtherte (28%) . Also, mte (28%)  and the te (28%)  predict lifespan . Free oflifespan . Mir-71 lifespan . Strikinrization .Among other methods, one of the most powerful machine learning approaches, particularly for image analyses, is the use of convolutional neural networks (CNN) , which aC. elegans were used. Each time point (day) has at least 30 worms, and all of them were anaesthetized before imaging. InceptionResNetV2-based architecture achieved a mean absolute error (MAE) of 0.96 day in the regression mode, and an accuracy of 57.6% in classification mode [Remarkably, CNN were recently used to predict lifespan in worms. In the first paper, a dataset of 913 images of ion mode . In anotion mode .C. elegans movement. To interpret classification results in a by-design fashion, we have accompanied classification CNN with a tandem segmentation CNN. For this, we devised a new U-Net-based architecture (HydraNet) for segmenting worms from the background and also segmenting the worm's body into anterior, mid-body and posterior parts. Interpretation of the classification results was achieved through the union of HydraNet segmentation and class activation maps generated using WormNet. The class activation maps analyses combined with body part segmentation in such tandem fashion allowed us to extract features responsible for lifespan prediction. Finally, using higher resolution segmented version of the C. elegans images, we verified our results in a higher expressive capacity residual CNN InceptionV3 accompanied by manual interpretation.Here we used the same dataset as in , 32, howC. elegans captured from day 1 of adulthood till death were used to develop our prototype platform [C. elegans we have reformulated the problem as a multi-class segmentation with one-hot encoded masks and similar U-Net-like architecture  as an activation function  on the test dataset for the day 3 adults fraction. The performance for the day 1 and day 2 images were slightly lower –2D with om noise . Our resass worm , 2F and ass worm , 2H fromNext, we used WormNet to classify long and short-lived worms. Similarly to movement classification, the WormNet performed better on day 3 adults sample reaching accuracy of 72%  on the test dataset, as compared to AUC ROC of 0.53 and 0.52 for day 2 and 1 respectively. The confusion matrix analysis suggested that the CNN underperformed in short-lived worms classifying –3C. NextTo verify these findings in an independent manner we have trained another lifespan classifier using the residual InceptionV3 architecture  accompanC. elegans being a classical model in aging research with more than 4000 papers published up to date, and the progress in robotics, the process of measuring C. elegans lifespan is still manual and laborious. However, new approaches are emerging like lifespan machine utilizing flatbed scanners to simultaneously assess the viability of a large population of worms on plates [Despite n plates . Anothern plates . The detn plates , 15. NucHere we worked with the dataset generated in Pincus lab , 15, andC. elegans lifespan [C. elegans morphology rather than the background of the images. Importantly, pretraining on the body-coordinate representations in [CNN were used before for regressing and classifying lifespan . The autlifespan . As mentlifespan . Increastions in  improvedIn addition to lifespan or movement classification based on young adults’ images, we also aimed to find features important for the prediction. As a prototype task we decided to determine which body part – anterior, mid-body or posterior part contains features influencing lifespan length the most. We designed HydraNet 3 and 4, new architectures based on U-Net and showed that they can successfully segment worm body parts achieving perfect Jaccard index values. Importantly, to develop a by-design interpretation approach we employed a tandem of biologically meaningful classification (lifespan and movement) yielding saliency through class activation maps , 35 and C. elegans in an automated setting, given a significant increase in image resolution and usage of body-coordinate representation. Non-labelled organs like pharynx or GFP-labelled entities could be segmented using HydraNets and assessed for their lifespan predictive ability using CAM approach and WormNet. It is tempting to speculate that akin to generative adversarial networks [C. elegans. This has a great potential to accelerate the pharmaceutical screening for anti-aging drugs. The development of the methodology will also be helpful to find and characterize new pathologies in C. elegans important for basic aging research. To make the code available to the research community we have deposited it on GitHub (https://github.com/ails-institute/DeepLongevity).This approach provides an avenue to the discovery of new important age biomarkers in networks , future https://github.com/ails-institute/DeepLongevity).All the source code for this work was implemented in Python version 3.6, Tensorflow versions 1.9.0 or 2.3.0  and KeraTraining of WormNet, and fine-tuning of InceptionV3 were performed on a desktop PC equipped with Intel Core i7-8700K CPU at 3.7 GHz and 32 GB of RAM as well as GeForce 1080 Ti GPU. Training of U-Net and HydraNet were performed using Google Collaboratory cloud GPU . Inference and analysis were performed using Google Collaboratory.https://github.com/ails-institute/DeepLongevity).WormNet used binary cross-entropy as a loss function and was trained using Adam optimizer with a starting learning rate of 0.005, beta 1 of 0.9, beta 2 of 0.999, epsilon of 1e-08, and decay of 0. A 30-fold data augmentation was performed for each epoch. U-Net and HydraNet were trained using Adam optimizer with a starting learning rate of 0.001 and default parameters. Dice loss function was used for the semantic segmentation task : Supplementary Figure 1


A simple, short, cheap, and reasonably sensitive and specific screening tool assessing both nutritional and non-nutritional risk factors for sarcopenia is needed. Potentially, such a tool may be the Mini Sarcopenia Risk Assessment (MSRA) Questionnaire, which is available in a seven-item (MSRA-7) and five-item (MSRA-5) version. The study’s aim was Polish translation and validation of both MSRA versions in 160 volunteers aged ≥60 years. MSRA was validated against the six sets of international diagnostic criteria for sarcopenia used as the reference standards. PL-MSRA-7 and PL-MSRA-5 both had high sensitivity (≥84.9%), regardless of the reference standard. The PL-MSRA-5 had better specificity (44.7–47.2%) than the PL-MSRA-7 (33.1–34.7%). Both questionnaires had similarly low positive predictive value . The negative predictive value was generally high for both questionnaires . PL-MSRA-5 had higher accuracy than the PL-MSRA-7 . Based on the results, the Mini Sarcopenia Risk Assessment questionnaire was successfully adopted to the Polish language and validated in community-dwelling older adults from Poland. When compared with PL-MSRA-7, PL-MSRA-5 is a better tool for sarcopenia risk assessment. Sarcopenia, which means a muscle failure, is a severe condition threatening healthy aging, increasing the risk of falls and injuries, physical disability, dependence, and even death . The diaTo the best of our knowledge, two questionnaires have been developed for the screening of sarcopenia—the SARC-F questionnaire  and MiniSarcopenia is a pluricausal condition. The useful clinical classification specifies two categories—primary and secondary sarcopenia ,25. PrimThe aim of our study was an elaboration of the Polish language version of the MSRA questionnaire and validation against six sets of international diagnostic criteria for sarcopenia: the European Working Group on Sarcopenia in Older People EWGSOP1) , the Ext , the ExTranslation and cross-cultural adaptation of the MSRA questionnaire was performed following the two phases World Health Organization (WHO) methodology for translating and intercultural adaptation of health questionnaires . The stuEach subject gave written informed consent before entering the study, which was conducted under the Declaration of Helsinki. The Bioethics Committee of the Poznan University of Medical Sciences, Poland approved the study protocol .We performed a face-to-face interview with each participant to assess sociodemographic factors  and clinical data (self-reported comorbidity and the number of drugs taken regularly).We used the Abbreviated Mental Test Score (AMTS) to assess subjects’ cognitive function . The full version of the Mini Nutritional Assessment (MNA) questionnaire was used to evaluate subjects’ nutritional status . SubjectIn addition to the MSRA questionnaire, we estimated the risk of sarcopenia with the SARC-F questionnaire  and its The validation process  was orgaThe MSRA questionnaire  has two A standardized forward-backward translation procedure was used to develop the Polish version of the MSRA . A physiAs a dish called ‘ragout’ is practically unknown in Poland, it was removed from the Polish version of the questionnaire. The word ‘ham’ was replaced with ‘cold meats’, as various types of sausages are popular in Poland. We also used the word ‘pulses’ instead of ‘legumes.’ In a satisfactory Polish version of MSRA-5 and MSRA-7, in the last question regarding weight loss in the last year, the second answer was expressed in the form: ‘no or ≤2 kg’ instead of ‘≤2 kg’.An English native speaker fluent in Polish, who did not know the MSRA questionnaire, performed the back translation. The same expert panel compared the original and back-translated versions, and found no substantial differences.We sent the back-translated questionnaires to Andrea Rossi, a co-author of the original MSRA, who accepted all changes and gave his consent to the Polish versions of the MSRA questionnaires. We obtained permission to use the prefix PL (for Poland) in the names of the evaluated questionnaires (PL-MSRA-7 and PL-MSRA-5), similarly to the prefix C in the Chinese versions (C-MSRA-7 and C-MSRA-5) . SupplemThe final Polish versions of MSRA-7 and MSRA-5 questionnaires were tested in 10 elderly subjects (five men and five women) with preserved mental function. The subjects were asked about perceived uncertainties concerning the questionnaire’s comprehension and cultural relevance. The inter-rater reliability and test-retest reliability of both MSRA versions was assessed by two independent researchers with a time interval of 2–4 weeks in 20 older subjects (10 men and 10 women). The number of respondents used in the inter-rater reliability and test-retest reliability assessment was based on the recommendations of the Special Interest Group on Sarcopenia of European Geriatric Medicine Society (EuGMS) for the validation studies of the SARC-F questionnaire , which wA clinical validation study was performed to assess the performance of the Polish version of the MSRA-7 and MSRA-5. Owing to the lack of a worldwide consensus of diagnostic criteria for sarcopenia, we used six sets of international diagnostic criteria as reference standards: 1) the EWGSOP1 ; (2) the the EWGS2) and the ALM/BMI index. We used the BIA method to assess muscle mass  and two indices of low muscle mass, i.e. the Appendicular Lean Mass (ALM) index  and the lower limb strength with the Chair Stand Test (CST). Physical performance was evaluated based on the 4-m usual gait speed test (UGS). The methodology of muscle mass, muscle strength, and physical performance has been described in detail in our previous publication . Table 2The PL-MSRA-7 and PL-MSRA-5 questionnaires  were validated against other functional and clinical measurements, such as age, HGS, CST, UGS, ALM, ALM/BMI, ALM index, BMI, MNA , ADL 3434, and It-test, the Cochran–Cox test, or Mann–Whitney test. Categorical variables (shown as numbers (percentage)) were analyzed with the χ2 test with the Yates correction if applicable.We used the STATISTICA 12.0 software  to perform statistical analysis. The distribution of data was checked with the Shapiro–Wilk test. The analysis of continuous data (shown as mean ± standard deviation (SD)) was performed with the Student’s The first phase of the study consisted in the assessment of reliability of translation and cross-cultural validation of the MSRA. The MSRA total score was considered to be a numerical variable, and the intraclass correlation coefficient (ICC) was calculated for the inter-rater and test-retest reliability. The level of agreement was defined, as follows: poor <0.5, moderate 0.50–0.75, good 0.75–0.90, and excellent >0.90 .The second phase of the study was aimed to assess the performance of the PL-MSRA-7 and PL-MSRA-5. To this end, five parameters were calculated: sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), and accuracy. The area under the curve (AUC) was assessed based on the receiver-operating characteristics (ROC) analysis. The diagnostic accuracy of the screening test based on AUC was defined, as follows: high >0.9, moderate 0.7 to 0.9, and low 0.5 to 0.7 .p < 0.05 was considered to be statistically significant.Relationships between PL-MSRA-5 and PL-MSRA-7  and other functional and clinical measurements were investigated while using the Spearman correlation coefficients. We used the following cut-off points for the strength of correlation: very good if a correlation coefficient of 0.81–1.00, good if 0.61–0.80, moderate if 0.41–0.60, and poor to fair if <0.40 . The difWe enrolled 160 elderly persons (aged 60–93 years), of whom 56% were women. p = 0.08). Over 1/3 of the study population were subjects aged ≥75 years. Approximately 50% of the study population were married. Twice as many women were unmarried as men (p < 0.001). The vast majority of subjects had at least secondary education. Every third participant was living alone; women lived alone three times more frequently than men (p < 0.001).Men were younger than women . The mean full MNA score in the total population indicated normal nutritional status. However, malnutrition or risk of malnutrition was diagnosed in 30% of the participants. No difference between men and women was found. Almost all of the participants were independent in activities of daily living assessed with the Katz scale. Men and women did not differ for the mean ADL and IADL scores. Based on face-to face interviews, the average number of chronic diseases was three, and regularly taken medicines was six. Participants most commonly declared hypertension (58.9%), chronic obstructive pulmonary disease (32.3%), cardiovascular disease (24.1%), dyslipidemia (20.3%), diabetes (18.4%), and osteoporosis/osteopenia (13.3%) (data available in 158/160 participants).Men were significantly higher (172.1 ± 6.4 vs. 156.8 ± 6.0 cm) and heavier (79.5 ± 14.0 vs. 66.8 ± 14.7 kg) than women, but the BMI was similar in both subgroups (26.8 ± 4.4 vs. 27.2 ± 6.1 kg/mp < 0.001). In contrast, both of the subgroups had similar lower limb strength. The usual gait speed was somewhat higher in men . Men had higher lean body mass and ALM index. p < 0.05) and SARC-CalF questionnaires .Women had significantly lower upper limb strength than men  in 10 men (mean age 74.6 ± 7.2 years) and 10 women (mean age 77.4 ± 7.5 years). We found an excellent agreement for the PL-MSRA-7 (0.910) and a good agreement for the PL-MSRA-5 (0.834).p < 0.001), indicating excellent agreement. The test-retest scores of the PL-MSRA-5 version only differed in one subject, which resulted in excellent test-retest agreement (95%) and ICC .One of the researchers (the physiotherapist) assessed the same 20 volunteers again at least two weeks (but no more than four weeks) later for the test-retest reliability check. For the PL-MSRA-7 questionnaire, the test-retest results differed in three cases; the test-retest agreement was 85%, and the test-retest intraclass correlation coefficient was 0.949  and a five-item (MSRA-5) version. The design of our validation study was based on recommendations by Bahat et al. , formulaUsing all of the currently available sets of international diagnostics criteria for sarcopenia, we found that both the full and shortened versions of the MSRA questionnaire have good sensitivity and are useful as a sarcopenia screening tool. Our findings are generally in accordance with previous studies ,8. PL-MSAs compared to the SARC-F questionnaire, the good point of the MSRA is the inclusion of nutritional items in screening for sarcopenia. Secondary sarcopenia may have multiple causes, with inadequate nutrition being among the most frequent. In our opinion, particularly important are MSRA questions concerning regular meals and intake of vegetable-based and animal-based proteins. It is well documented that both nutritional caloric intake and adequate amount, quality, and distribution of dietary proteins are important for the restraining of muscle mass loss ,45,46,47Another important item in the MSRA questionnaire is weight loss in the past 12 months, and weight loss exceeding 2 kg is regarded as a risk factor for sarcopenia. Sarcopenia is particularly frequent in elderly subjects with unintentional weight loss . HoweverThe use of the BIA method for muscle mass measurement is an important limitation to our study. While it is well-known that most precise methods of muscle assessment are magnetic resonance imaging (MRI), computed tomography (CT), or dual energy X-ray absorptiometry (DEXA), these methods are difficult to use in larger populations due to their high cost and technical requirements . DEXA isThe assessment of muscle mass with non-invasive BIA method can be repeated without restrictions. The BIA method is simple and cheap, and the equipment is portable, which enables assessment in patient’s place of living.A good point of our study is the use of all six currently available sets of international diagnostic criteria for sarcopenia as reference standards for MSRA validation: European Working Group on Sarcopenia in Older People 1 (EWGSOP1) , the ExtThe Mini Sarcopenia Risk Assessment questionnaire was successfully adopted in Polish and validated in community-dwelling older adults from Poland. Compared with PL-MSRA-7, PL-MSRA-5 is a better tool for sarcopenia risk assessment. As only three validation analyses (including the current one) have been performed so far, further studies are needed to demonstrate the MSRA is a useful screening tool for sarcopenia in clinical practice. Such analyses should preferably involve other settings  and comparisons between the MSRA and SARC-F questionnaires.


Non-invasive diagnostic and prognostic biomarkers for urinary bladder cancer (BC) are eagerly awaited, since these could improve patient’s quality of life and reduce resources and costs for the health care system. The PD-1/PD-L1 axis is an important immune escape mechanism for tumors, and both high tissue expression and soluble levels of PD-L1 have been shown to be associated with worse prognosis for patients with various tumor. The knowledge about soluble PD-L1 in body fluids such as serum and urine in patients with BC is sparse, and more studies are needed to investigate its role as a biomarker for the disease. In the present study, we show that high serum PD-L1 levels are associated with aggressive disease and development of metastatic disease in BC patients.p = 0.038 and p = 0.61, respectively). Significantly higher serum sPD-L1 levels were found in patients with muscle invasive disease and metastatic disease, compared to patients with non-muscle invasive BC and non-metastatic disease (p < 0.05). There was also a trend for higher urine sPD-L1 levels in patients with metastatic disease, compared to patients with non-metastatic disease (p = 0.05). The results from this study suggest that sPD-L1 in serum, but not in urine, could be a potential prognostic biomarker for patients with BC.Soluble PD-L1 (sPD-L1) levels have been identified as a potential biomarker for various cancers, but its diagnostic and prognostic value in urinary bladder cancer (BC) remains to be fully elucidated. In this study, we investigated sPD-L1 levels in serum and urine samples from 132 patients with BC and compared them to 51 patients with hematuria (controls). The levels of sPD-L1 in serum and urine were determined using ELISA. Soluble PD-L1 could be detected in 99.5% of the serum samples and 34.4% of the urine samples. Patients diagnosed with BC had significantly higher urinary levels of sPD-L1, compared to controls, however no difference were found in serum sPD-L1 levels ( Urothelial bladder cancer (BC) is the ninth most common cancer worldwide with an estimation of 549,000 new diagnoses in 2018, and each year 200,000 people die due to the disease . PatientIn order to survive and progress, tumor cells needs to evade destruction by the immune system. One mechanism for this is to deregulate immune checkpoints, which are involved in a plethora of pathways crucial for maintaining self-tolerance and for modulating the duration and amplitude of immune responses. One such immune checkpoint is the programmed cell death ligand 1 (PD-L1), which is a ligand to the co-inhibitory receptor programmed cell death 1 (PD-1). Binding of PD-L1 to PD-1 results in inhibition of the T-cell mediated immune response, which could result in tumor cells escaping immune surveillance ,7. The eBoth PD-1 and PD-L1 exist in soluble forms, namely soluble PD-1 (sPD-1) and soluble PD-L1 (sPD-L1) . SolublePatients were recruited from the BLAdder cancer Blood and Urine Study (BLABUS), a prospective cohort study consisting of patients with macroscopic hematuria who were referred to the Department of Urology at the University hospital of Örebro for suspicion of bladder cancer. The vast majority of the patients underwent computed tomography urography, and an outpatient cystoscopy. Patients with visual suspicion of a bladder tumor at cystoscopy or urography thereafter underwent a transurethral resection of the bladder tumor (TUR-B) under general anesthesia. The diagnosis of BC was confirmed by a histopathological examination of the resected tissue samples. Tumor stage was defined according to the 2009 TNM classification of BC, and tumor grade was designated according to the WHO 2004 and WHO 1999 grading schemes. Patients without a history of BC and with a normal cystoscopy and urography were included as controls. Clinical data were extracted from medical records until 1 May 2019. Cause of death was defined as cancer specific if the patient had metastatic BC at time of death, and unrelated to BC if no signs of metastatic BC were detected at time of death. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the ethics committee in Uppsala/Örebro region .g for 10 min at 4 °C. Both urine and serum samples were stored at −80 °C until analyses were performed. All samples were analyzed retrospectively, and samples from cases and controls were processed simultaneously. At inclusion, blood and urine samples were obtained from all patients, and were collected prior to any cystoscopy or treatment of the patient. Urine samples were centrifuged at 2000 rpm for 10 min, and the serum was obtained by centrifugation at 3000× Soluble PD-L1 in serum and urine were measured by a commercially available ELISA for PD-L1, according to the manufacturer´s instructions . Control samples with known concentrations of PD-L1  were included in each ELISA . The minimum detectable level of sPD-L1 in the ELISA was 4.52 pg/mL, and the detection range was 25.0–1600 pg/mL. The optical density was measured using a Multiskan Ascent plate reader  at 450 and 540 nm. 2-values above 0.9 were considered as acceptable. Samples were measured in duplicates, and for each sample the coefficient of variation  was calculated. The acceptable range of CV was 0–10%, samples with higher CV values were analyzed one more time. Blanks and standards were assayed according to the manufacturer´s instructions. The mean values of Absorbance vs. Concentrations were plotted and a 4-Parameter Logistic (4PL) non-linear regression model fit was applied, and Rp < 0.05.In order to test the data for normality, a Shapiro-Wilks test was used. A Mann-Whitney U test or Kruskal-Wallis test were subsequently used to compare sPD-L1 levels between groups of patients. The urinary levels of sPD-L1 were dichotomized into absent or present, based on the detection level of sPD-L1 in the analysis method, and a Chi-square test was performed to test for association between dichotomized urinary sPD-L1 and cancer. Chi-square or Fisher´s exact tests were performed to test for associations between categorical variables. A Spearman correlation test was used to test for correlation between continuous data variables. Levels of sPD-L1 were subsequently divided into low or high, based on the median level of sPD-L1 among cases. The Kaplan-Meier method was used to estimate survival probabilities and the log-rank test was used to compare groups. A logistic regression model was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) of the association between sPD-L1 levels and metastatic disease. All analyses were performed in SPSS version 22.0 . A statistical significance was considered at A total of 132 BC patients and 51 controls were recruited between 1 October 2016 and 31 March 2019. Compared to controls, patients diagnosed with BC were significantly older at inclusion, they were more commonly current or former smokers, and of male sex. No difference in body mass index (BMI) at the time of inclusion were seen between cases and controls . Of the p = 0.002). No correlation between sPD-L1 in serum and BMI, age, or smoking status were found. Soluble PD-L1 could be detected in 182 of 183 (99.5%) of the available serum samples and the median concentration was 95.2 pg/mL (65.2–214.4 pg/mL). Men had significantly higher levels of sPD-L1, compared to women . No significant difference in serum levels of sPD-L1 between cases and controls were found . When adjusting the model for tumor grade (low vs. high) and stage, this association was diminished, and no longer statistically significant . Patients that died during follow-up  had significantly higher serum sPD-L1 levels compared to patients who survived . A survival analysis showed that patients with sPD-L1 levels above median (94.0 pg/mL) had shorter overall survival time (19.6 months), compared to patients with sPD-L1 levels below the median value . Furthermore, no correlations were found between sPD-L1 in urine and BMI, age, or smoking status. Soluble PD-L1 could be detected in 56 of 163 (34.4%) of the available urine samples, and the median concentration was 74.2 pg/mL (57.5–669.2 pg/mL). No correlation between serum sPD-L1 and urinary sPD-L1 levels were found . No associations between sPD-L1 levels in urine and pT-stage and grade (low vs. high) were evident , however patients who were diagnosed with metastatic disease or who developed distant metastases during follow-up had higher levels of urinary sPD-L1, compared to patients without metastatic disease (p = 0.05). No association between sPD-L1 levels in urine and all-cause mortality was found (p = 0.09).Patients with BC had higher sPD-L1 levels in urine compared to controls  compared to patients with low stage disease (T0–T2), although they found no association between sPD-L1 and lymphatic or visceral metastases . SupportEven though there have been therapeutic breakthroughs for BC with the use of antibodies targeting PD-1 or PD-L1, there are still many questions that needs to be answered. One of the most important questions is why only a portion of the patients responds to immunotherapy. Thus, there is a demand for biomarkers that can identify responders versus non-responders prior to applying immunotherapy. Levels of sPD-L1 have previously been associated to treatment response to the immune checkpoint inhibitor Nivolumab (anti-PD-1) in patients with e.g., lung cancer . FurtherA strength with the current study is the choice of controls, which reflects a clinical setting where the major aim is to separate between patients presenting with hematuria due to BC or other causes. We have also investigated the levels of sPD-L1 in both serum and urine samples from these patients. The study also has some limitations, mainly the small cohort size and the short follow-up time of the patients . Another limitation of this study is that the tissue expression of PD-L1 were not measured in these patients, thus making it impossible to compare mPD-L1 expression to sPD-L1 levels in serum and urine. Even though the study was underpowered for some of the analyses, the results suggest a potential value of serum sPD-L1 as a prognostic biomarker for BC, which motivates further investigations using larger study cohorts with longer follow-up time. Our results showed that sPD-L1 can be detected in both serum and urine samples in BC. Patients with BC had higher urinary sPD-L1 levels than controls, but since only a third of the samples had detectable levels it appears like urinary sPD-L1 is unsuitable as a biomarker for BC. High sPD-L1 levels, mainly in serum, were found to be associated with a more aggressive BC, as indicated by pT-stage and metastatic disease, and could thus be a potential prognostic biomarker for the disease. Further prospective and longitudinal studies are needed to assess serum sPD-L1 as a biomarker for monitoring of BC patients.


Buxus sempervirens L. is a common ornamental plant in southern and central Europe, and has been used ethopharmacologically against a wide variety of diseases due to it containing nor-triterpene alkaloids of the nor-cycloartane type. Recently, we demonstrated the interesting antiprotozoal potential of some of these compounds. To characterize the temporal variability in the alkaloid profile of two different varieties and their leaves and twigs, 30 different extracts of B. sempervirens were evaluated by Ultra High Performance Liquid Chromatography/positive Mode-Electrospray Ionization Quadrupole Time-of-Flight-Tandem Mass Spectrometry (UHPLC/+ESI-QqTOF-MS/MS). The analytical profiles were thoroughly investigated by various methods of multivariate data analysis (MVDA). A principal component analysis (PCA) model elucidates the seasonal variation in the phytochemical composition of B. sempervirens var. arborescens and suffruticosa along with differences between the varieties. Analysis of a volcano plot illustrated the differences between the two organs, the leaf and twig. Eighteen compounds were highlighted by the models as constituents of the plant characteristic for a season, variety or organ. These compounds were dereplicated based on their chromatographic and +ESI-QqTOF-MS and –MS/MS data. In addition, mass spectral fragmentation pathways for already known alkaloids as well as new natural products could be postulated for the first time. In conclusion, the MVDA models give detailed information on the temporal variability in the alkaloid profile of two different varieties and their organs (leaf vs. twig) of B. sempervirens. Thus, the results of this study allow, e.g., the identification of characteristic compounds for the different varieties, plant organs, seasons, and the optimal harvesting time for the isolation of particular Buxus-alkaloids of interest for subsequent studies. Buxus sempervirens L. (Buxaceae), has been used as a medical plant to cure, amongst other diseases, rheumatism and malaria 2+ < [M + H]+), so that the other one should be part of an amide group. The fragments m/z 444 [M + H-(CH3)2NH]+ and 323 [444-C6H5CONH2]+ consistently indicated the neutral loss of a dimethylamino group and a benzamide moiety. To the best of our knowledge, no Buxus-alkaloid has been described so far with these features represented by the putative structure in Compounds by LC/MS  and the -6 and 7 . It coul30  and 32 (11.94 min: 366.280 m/z) were highlighted as unique features. Compound 31 displayed a characteristic fragmentation pattern  + and 325 [446-C6H5CONH2]+ indicated a substitution with a dimethylamino group and a secondary benzamide. The complete fragmentation pathway is shown in For compound by LC/MS . Due to s/amides . The fra36 (11.22 min: 384.327 m/z), 37 (12.81 min: 285.162 m/z) and 38 (12.54 min: 384.259 m/z) were present in the twigs in significantly higher quantities than in the leaves studied 2NH)]+ resulted from the neutral loss of a dimethylamino group (−45 Da), while the subsequent fragment at m/z 321 [339-H2O]+ indicated the presence of a hydroxyl group. The core fragment m/z 295 in the related +ESI-QqTOF MS/MS spectrum indicated a Buxus-alkaloid with a double bond between C-6 and C-7  to 1 mm mesh size. Of each sample, 3.5 g of ground drug material was extracted with 35 mL of dichloromethane (DCM) on a magnetic stirrer  for 30 min at room temperature. The extraction was repeated twice with 35 mL of the same solvent and the entire extract was evaporated to dryness on the rotary evaporator .A total of 30 extracts  were obtained and analyzed by LC/MS .B. sempervirens var. arborescens, leaves) was chosen as a quality control (QC) and analyzed after every 20 sample injections. Likewise, a blank run (MeOH) was carried out after 20 injections but in alternating order with the QC so that either QC or Blank were analyzed after every 10th sample injection.Each extract was dissolved in methanol (MeOH) at a concentration of 10 mg/mL for recording a fingerprint chromatogram (full scan MS) by UHPLC/+ESI-QqTOF-MS  using the same method and parameters as previously described . As an iThe pretreatment of LC/MS data and the creation of a descriptor table (“bucket table”) were carried out with the software ProfileAnalysis 2.1  in the same way as in our previous study . A valueThe data of the bucket table (1200 bucket variables) were mean-centered by default in ProfileAnalysis before the models were created. In addition, three different scaling methods  and the use of the unscaled data (none) for model calculation were tested  and 2.3.Based on the bucket table, a t-test was carried out in the ProfileAnalysis software  in order to find constituents distinguishing leaves from twigs. The fold change, which indicates the concentration change of a substance or a metabolite in different samples, and the value count, which points out in how many samples the bucket are detectable, were determined.2(fold change)| ≥ 2.32 for leaf and |log2(fold change)| ≥ 2 for twig) and a low p-value  ≥ 1.3)) [The unique features  were taken directly from the t-test result table, while the compounds that showed significant differences in content between the twig and leaf extracts were highlighted in the volcano plot. Buckets with a high fold change (|log ≥ 1.3))  were conB. sempervirens var. arborescens and suffruticosa. The results will allow the selection of the right plant variety and optimal harvesting time to obtain certain alkaloids that are, e.g., of interest because of a particular biological activity. The results of the MVDA of the complex dataset by principal component and t-test/volcano plot analysis yield a holistic picture of differences in the main constituents of the two varieties and their leaves and twigs. In addition to this valuable information, these detailed comparative studies also provide interesting data on the seasonal changes in secondary metabolites in the plants studied. Thus, marker substances for the different varieties such as N-benzoyl-O-acetylbuxadine-E (23) for B. sempervirens var. arborescens L. and Nb-dimethylcycloxobuxoviricine (12) for B. sempervirens var. suffruticosa L. could be identified. Similarly, leaves and twigs can be distinguished by the occurrence of buxruguloid-B (30) and compound 31 , respectively. It is to be noted that, based on detailed LC/MS analysis, +ESI fragmentation pathways of various known and several putative new Buxus-alkaloids were established here for the first time, which will enable the rapid dereplication of these natural products in future studies.The present study, for the first time, shows the main chemical differences between the investigated varieties,